The Over-Irradiation Metabolite Derivative, 24-Hydroxylumister-ol3, Reduces UV-Induced Damage in Skin

The hormonal form of vitamin D3, 1,25(OH)2D3, reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D3 to produce “over-irradiation products” such as lumisterol3 (L3). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L3 to produce three main derivatives: 24-hydroxy-L3 [24(OH)L3], 22-hydroxy-L3 [22(OH)L3], and 20,22-dihydroxy-L3 [20,22(OH)L3]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L3, in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L3 or 1,25(OH)2D3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH)2D3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L3 provided photoprotection against UV damage similar to that of 1,25(OH)2D3.


Introduction
While sunlight clearly has many positive benefits, the detrimental effects of sun exposure on skin were first proposed by Paul Gerson Unna in 1894 [1]. Studies since then have Figure 1. Biosynthesis and metabolism of vitamin D and over-irradiation products after UV radiation. When 7-dehydrocholesterol in skin absorbs UVB, it is converted to pre-vitamin D3. Pre-vitamin D3 thermo-isomerizes into vitamin D3 at body temperature. Vitamin D is converted in the liver and other tissues, including skin, to 25-hydroxyvitamin D3 by CYP2R1/CYP27A1 enzymes and then to the biologically active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in the kidney and other tissues including skin by CYP27B1. Exogenous vitamin D can be acquired through food, such as fish or supplements. Continuous UVB exposure photo-isomerizes pre-vitamin D3 to produce what used to be considered relatively biologically inactive "over-irradiation products" such as lu-misterol3 (L3), tachysterol3, and other compounds. CYP11A1 is a cytochrome P450 enzyme found in skin which catalyzes L3 to 22(OH)lumisterol3, 24(OH)lumisterol3, and 20,22(OH)2lumisterol3 as the major products. Green color shows classic vitamin D compounds. Blue color shows over-irradiation products and metabolites.

Cell Cultures and Skin Explants
Skin samples for keratinocyte culture or skin explant culture were harvested after obtaining written informed consent from subjects or their parents/guardians, with Figure 1. Biosynthesis and metabolism of vitamin D and over-irradiation products after UV radiation. When 7-dehydrocholesterol in skin absorbs UVB, it is converted to pre-vitamin D 3 . Pre-vitamin D 3 thermo-isomerizes into vitamin D 3 at body temperature. Vitamin D is converted in the liver and other tissues, including skin, to 25-hydroxyvitamin D 3 by CYP2R1/CYP27A1 enzymes and then to the biologically active vitamin D hormone, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), in the kidney and other tissues including skin by CYP27B1. Exogenous vitamin D can be acquired through food, such as fish or supplements. Continuous UVB exposure photo-isomerizes pre-vitamin D 3 to produce what used to be considered relatively biologically inactive "over-irradiation products" such as lumisterol 3 (L3), tachysterol 3 , and other compounds. CYP11A1 is a cytochrome P450 enzyme found in skin which catalyzes L3 to 22(OH)lumisterol 3 , 24(OH)lumisterol 3 , and 20,22(OH) 2 lumisterol 3 as the major products. Green color shows classic vitamin D compounds. Blue color shows over-irradiation products and metabolites.

Cell Cultures and Skin Explants
Skin samples for keratinocyte culture or skin explant culture were harvested after obtaining written informed consent from subjects or their parents/guardians, with approval from the University of Sydney Human Ethics Committee (Reference number 2015/063).

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Keratinocytes: Keratinocytes were grown out from skin fragments [75] and cultured as previously described with minor modifications [76], in keratinocyte growth medium (KGM), which contained minimum essential medium Eagle (M0518, Sigma-Aldrich, St. Louis, MO, USA), 0.02 M sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium pyruvate, and 25 mM HEPES (ThermoFisher Scientific, Waltham, MA, USA) in MilliQ water at pH 7.2 (Millipore SAS, Molsheim, France). Human primary keratinocytes were cultured in KGM containing 5% (v/v) fetal calf serum (FCS) and the following supplements: 5 µg/mL transferrin, 0.4 ug/mL hydrocortisone, 1 × 10 −10 M cholera toxin, 10 ng/mL Epidermal Growth Factor (EGF), 5 µg/mL insulin (all from Sigma-Aldrich, St. Louis, MO, USA), and 2 × 10 −11 M 3,3,5-triiodo-L-thyronine sodium salt [77]. Keratinocytes from passages 2-5 from at least two independent donors were used in all experiments. The keratinocyte culture media was changed to media without EGF and cholera toxin for 24 h before experiments to allow cells to become quiescent [78]. • Skin explants: Human skin explants were collected from consenting patients undergoing elective surgery at private hospitals in Sydney, Australia, and processed as previously described [56]. In brief, ice-cold sterile phosphate buffered saline (PBS) was used to transport the skin back to the laboratory. The skin was processed under aseptic conditions within 4 h of surgery. The skin was briefly washed with 4% chlorhexidine gluconate solution (Sigma-Aldrich, St. Louis, MO, USA) and then thoroughly washed with ice-cold PBS. Subcutaneous fat and debris were trimmed off to leave the epidermis and dermis only, for the study. The skin samples were dissected into 4 mm pieces with a punch biopsy tool with five biopsies prepared for each treatment. The skin samples were then prepared for UV irradiation as described below.

Solar-Simulated UV Irradiation
UV irradiation was provided by an Oriel Sol1A TM 94042A 450 W solar simulator (Newport Corporation, Irvine, CA, USA) with an atmospheric attenuation filter to eliminate UVC (<290 nm). It was calibrated using the OL756 spectroradiometer (Gooch & Housego, Melbourne, FL, USA), and an IL 1700 broadband radiometer (International Light Technologies, Peabody, MA, USA) was used immediately prior to experiments to determine the UVB output, from which a calculated dose of solar-simulated UV (ssUV) was determined. This dose of UV did not increase caspase activity or decrease the total DNA and equated to approximately 4 min of sunlight at noon in October in Sydney, Australia [79]. The spectral output of this lamp as used for these experiments has been published [ Figure S6 in [79]].

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Keratinocytes: Immediately prior to UV irradiation, the medium was replaced with irradiation buffer, Martinez solution containing 10 mM D-glucose (Sigma-Aldrich, St. Louis, MO, USA) without phenol red. For all keratinocyte experiments, the ssUV irradiation energy level was 400 mJ/cm 2 UVB and 3600 mJ/cm 2 UVA (4000 mJ/cm 2 ) [53,79]. • Skin explants: Skin biopsies were placed in ice-cold sterile colorless Martinez buffer with the epidermis facing up in a volume that was just enough to surround the tissue without submersion, for UV radiation [56]. A single dose of ssUV at 20 J/cm 2 was delivered to the skin explant samples. Sham/non-irradiated keratinocytes or skin were subjected to similar procedures but not irradiated.

1,25( OH) 2 D 3 and 24(OH)L 3 Treatments
The 1,25(OH) 2 D 3 was from Sapphire Bioscience Pty Ltd., (Sydney, Australia). The 24(OH)L 3 was enzymatically synthesized using recombinant CYP11A1 and purified using procedures as previously described [72,80]. These compounds were solubilized in spectroscopic grade ethanol (Merck, Darmstadt, Germany) and added to cultures in 0.1% (v/v) ethanol. The ethanol vehicle 0.1% (v/v) was used as a control. The concentration of these compounds was determined with a Nanodrop ND-1000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) immediately prior to use.

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Keratinocytes: Immediately after UV irradiation (or sham irradiation), the irradiation buffer was replaced with supplement-free keratinocyte growth medium (KGM) containing vehicle, 0.1% (v/v) ethanol, or treatments at the concentrations as indicated.

Immunohistochemistry
The current study used an antibody detection method for thymine dimers as an index of total CPDs and antibody detection of 8-OHdG followed by image analysis. These methods produced similar results to those that detected CPDs or 8-OHdG by a specific endonuclease followed by a Comet assay [54,76]. CPDs consist of thymine-thymine, thymine-cytosine, cytosine-thymine, or cytosine-cytosine photoproducts [81]. Using a direct assay of a combination of liquid chromatography and tandem mass spectrometry to determine the different fractions of dimer photoproducts after UV, thymine dimers were found to be the most abundant, eightfold higher than other photolesions and correlated with the number of other dimers [9,82]. Thus, thymine dimers are routinely used as a measure of the total CPDs.

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Keratinocytes: CPDs and 8-OHdG: At the end of the incubation period after UV exposure (3 h or the period mentioned for the experiment), the keratinocytes were rinsed with PBS after incubation, and the keratinocytes were fixed with ice-cold 100% methanol (Sigma-Aldrich, St. Louis, MO, USA) at −20 • C for 10 min. Cells were extensively washed with MilliQ water and air dried overnight prior to immunohistochemistry, which was carried out as previously described [55], with minor modifications. Endogenous peroxidase activity was blocked by 1% (v/v) H 2 O 2 (ThermoFisher Scientific, Waltham, MA, USA) in PBS for 5 min followed by two MilliQ water washes. Antigen retrieval was carried out in several steps. Nuclear DNA was denatured with 70 mM NaOH (Sigma-Aldrich, St. Louis, MO, USA) in 70% ethanol for 2 min. Proteolytic digestion was achieved with 1 µg/mL Proteinase K (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 mM CaCl 2 for 5 min for CPD detection and 10 min for 8-OHdG detection followed by two MilliQ water washes. Non-specific antibody binding was inhibited by treating cells with 50% (v/v) horse serum (Sigma-Aldrich, St. Louis, MO, USA) in PBS (blocker) for 1 h. The cells were incubated with primary antibody for 60 min at room temperature, either mouse monoclonal anti-thymine dimer at 5 µg/mL (Sigma-Aldrich, St. Louis, MO, USA) for CPDs or primary mouse monoclonal IgG 2b anti-8-hydroxy-guanosine antibody (Santa Cruz Biotechnologies, Dallas, TX, USA) for 8-OHdG at 2.5 µg/mL or an equivalent concentration of isotype control, followed by three TBS-T washes. The cells were incubated with biotinylated goat anti-mouse IgG secondary antibody (ThermoFisher Scientific, Waltham, MA, USA) diluted to 1:500 in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 15 min followed by three TBS-T washes. Horseradish peroxidase (HRP)-Streptavidin Conjugate (Invitrogen, Waltham, MA, USA) diluted 1:150 in TBS-T was added for 15 min followed by three TBS-T washes. The HRP substrate Diaminobenzidine (DAB) (Enhanced Liquid substrate System for Immunohistochemistry, Sigma-Aldrich, St. Louis, MO, USA) was added for 5 min followed by two MilliQ water washes. Isotype antibodies were run as controls for all experiments, but as previously published, the isotype controls resulted in minimal staining [83]. The coverslips were mounted on glass slides with entellen rapid mounting medium (Merck, Darmstadt, Germany) for image analysis [55].

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Skin explants: After 3 h incubation, skin samples were fixed in 10% (v/v) neutral buffered formalin overnight, then paraffin-embedded and sectioned. The paraffin-embedded sections were first deparaffinized in xylene (ThermoFisher Scientific, Waltham, MA, USA), then hydrated by passing the slides through graded alcohol. Antigen retrieval was carried out by treating sections with citrate buffer, pH 6, at 95 • C for 30 min as previously described [56].
CPDs or 8-OHdG: Only for 8-OHdG staining, sections were incubated with RNase A (AMRESCO, Cleveland, OH, USA) at 37 • C for 30 min to remove 8-OHdG that would otherwise be detected in cellular RNA [84], followed by two TBS-T washes. For both lesions, sections were treated with 2 N HCl (Sigma-Aldrich, St. Louis, MO, USA) in 70% ethanol for 15 min at room temperature to denature DNA and further expose damaged sites. Following aspiration, the residual acid solution was neutralized with 50 mM Tris-HCl (pH 7.5) for 15 min at room temperature, followed by two further TBS-T washes. Sections were then blocked with 10% (v/v) horse serum in PBS, pH 7.2, for 60 min at room temperature, then incubated with primary antibody for 60 min at room temperature, either mouse monoclonal anti-thymine dimer at 10 µg/mL for CPDs or primary mouse monoclonal IgG 2b anti-8hydroxy-guanosine antibody at 2.5 µg/mL for 8-OHdG or an equivalent concentration of isotype control, followed by three TBS-T washes.
XPC or XPA: Following the antigen retrieval, the XPC and XPA sections were blocked with avidin and biotin from the Dako Cytomation Blocking System (Dako, Glostrup, Denmark) for 10 min at room temperature with a brief rinse in between treatments, followed by blocking with 10% (v/v) horse serum in PBS for 60 min at room temperature. The sections were incubated with primary antibodies overnight at 4 • C, either with mouse monoclonal IgG 2a XPC at 2.5 µg/mL, mouse monoclonal IgG 1 XPA antibody at 4.0 µg/mL (both from Santa Cruz Biotechnology, Dallas, TX, USA), or an equivalent concentration of isotype control, followed by three TBS-T washes.
Detection of antibody binding: This was carried out for all sections with the Dako LSAB Plus REAL Detection System Kit with AEC chromogen staining, using the methods provided by the manufacturer (Dako, Glostrup, Denmark). The slides were mounted with DPX (Sigma-Aldrich, St. Louis, MO, USA) and coverslipped.

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Keratinocytes: Bright-field images were acquired on the Olympus stereo investigator scope (MBF Bioscience, Williston, VT, USA) or on the Zeiss AxioScan-Z1 slide scanner (Zeiss Microscopy, Oberkochen, Germany) at the Bosch Advanced Microscopy Facility (University of Sydney). All images were taken at 20× magnification, and the immunohistochemical images were analyzed using ImageJ software. Using this program, images from an individual experiment were thresholded to the same value. The regions of interest (ROI) were randomly selected. The means and standard errors of the mean (SEMs) from three to five ROI per coverslip of each treatment were calculated and graphed. Staining and image analysis for CPDs and 8-OHdG produced similar results to those obtained using endonuclease detection of the lesion, followed by Comet assay [54,76]. • Skin explants: Bright-field images were captured using the Zeiss Axio Scan (Zeiss Microscopy, Oberkochen, Germany). For each section, images of the whole skin section were taken at 20× magnification. All images were analyzed using MetaMorph imaging software (Molecular Devices Corporation, San Jose, CA, USA), whereby the epidermal area was isolated and thresholded specific for positive nuclei in this region. This software then automatically calculated the positive nuclei as a percentage of the total epidermal area.

siRNA Transfection
Plated keratinocytes were transfected with 50 nM siRNA directed at XPC (siXPC), XPA (siXPA), or a control non-directed sequence (siCTRL) (all from Santa Cruz Biotechnologies, Dallas, TX, USA), following combination with a lipid-based transfection reagent (Santa Cruz Biotechnologies, USA) in Opti-MEM™ serum-free media (ThermoFisher Scientific, MA, USA) according to the manufacturer's instructions and as previously described [85]. The cells were incubated overnight, and on the following day, an equal volume of 10% (v/v) FCS in KGM without EGF and cholera toxin was added to deliver a final concentration of 5% (v/v) KGM without EGF and cholera toxin. The cells were further incubated overnight under standard culture conditions prior to irradiation and treatment.

Western Blot
Keratinocytes were plated directly in six-well plates in KGM with supplements. Following the indicated treatments, the cells were lysed and subjected to Western blot as previously described [79] with minor modifications. The transfer of protein to a nitrocellulose membrane (AmershamTM Protran 0.45 lm NC; GE Healthcare) was achieved at 25 V overnight at 4 • C. The membrane was then blocked with 5% (w/v) BSA in TBS-T at pH 7.2 for 1 h before being incubated with mouse monoclonal IgG 2a XPC (Santa Cruz Biotechnologies, CA, USA) at 1 µg/mL in 5% (w/v) BSA in TBS-T, overnight at 4 • C. The following day, the membrane was washed with TBS-T three times and incubated with secondary goat anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) in 5% (w/v) BSA in TBS-T for 1 h at room temperature. After incubation, the membrane was washed three times with TBS-T before adding chemiluminescence substrate (Millipore SAS, Molsheim, France) for band detection. The bands were imaged with the ChemiDocTM imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at the Bosch Research Institute (University of Sydney), and densitometry was carried out using ImageJ software (version 1.50).

Unscheduled DNA Synthesis
This was carried out as previously described [79]. In brief, EDU (5-ethynyl-2 -deoxyuri dine) incorporation into nuclear DNA was detected using the iClick TM EdU Andy Fluro 488 Imaging Kit (GeneCopoeia Inc, Rockville, MD, USA). The incorporation of this thymidine analog in a punctate pattern, in keratinocytes cultured without growth factors EGF and cholera toxin, measured unscheduled DNA synthesis rather than DNA replication [86]. Densitometry was performed using ImageJ software. The stained cells were manually counted from the images taken with a Zeiss LSM 510 Meta confocal microscope (Zeiss Microscopy, Oberkochen, Germany). The fluorescence intensity was measured in cells that were above the detection threshold and considered "UDS positive", and fluorescence images were acquired by confocal microscopy and analyzed using ImageJ software [79].

Seahorse Energetics
An XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent, CA, USA) at Bosch Research Institute, University of Sydney, was used immediately following irradiation of cells to measure ECAR, as previously described [79].

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In vitro: Three independent experiments with triplicates per each treatment group were performed for each study with similar results, using keratinocytes from different donors. Unless otherwise indicated, the analyses were carried out by ANOVA with Tukey multiple comparisons post-test (GraphPad Prism statistical program) (CA, USA). • Ex vivo: Three independent experiments were performed for each study with skin from different donors with similar results. Comparisons between treatments were made by one-way ANOVA followed by Sidak's multiple comparisons test, using the GraphPad Prism statistical program (CA, USA), unless otherwise stated. The data in the graphs represent the mean + SEM, unless otherwise stated.

24(OH)L 3 Increased Unscheduled DNA Synthesis Similar to 1,25(OH) 2 D 3 in UV-Irradiated Human Primary Keratinocytes
To examine whether these vitamin-D-related compounds increased DNA repair, we tested unscheduled DNA synthesis (UDS). UV-irradiated keratinocytes or non-irradiated control cells were treated immediately after with vehicle, 1,25(OH) 2   DNA repair requires energy [38,87]. Exposure to UV irradiation reduced ATP levels in keratinocytes ( Figure 3F). Treatment with 24(OH)L 3 or 1,25(OH) 2 D 3 immediately after UV increased the ATP levels measured 1.5 h after UV exposure in keratinocytes (p < 0.05) compared with the UV vehicle. Significant differences were not observed between the different concentrations of either 24(OH)L 3 or 1,25(OH) 2 D 3 . Significant changes in ATP levels were not observed in non-irradiated cells treated with either 24(OH)L 3 or 1,25(OH) 2 D 3 compared with the non-UV vehicle ( Figure 3F).  We previously reported that the oxygen consumption rate (a measure of oxidative phosphorylation) decreased in UV-exposed keratinocytes [79]. Thus, in order to determine the source of the energy in these irradiated and treated keratinocytes, the extracellular acidification rate (ECAR), a measure of glycolysis, was measured using Seahorse XF analysis ( Figure 4A). The results indicated that there were no significant changes in the non-irradiated or irradiated keratinocytes over the first 25 min of equilibration after sham or UV exposure prior to the injection of treatments. Significant increases in the ECAR, indicating increased glycolysis, were observed in UV-irradiated, 24(OH)L 3 -treated cells (p < 0.01) and in UV-irradiated, 1,25(OH) 2 D 3 -treated cells (p < 0.05) ( Figure 4B) compared with UV-irradiated vehicle-treated keratinocytes.

Reductions in UV-Induced CPDs and 8-OHdG by 24(OH)L 3 or 1,25(OH) 2 D 3 Were Abolished in the Presence of the Glycolysis Inhibitor (2-Deoxy-D-glucose) but Not by the Oxidative Phosphorylation Inhibitor (Oligomycin) in Human Primary Keratinocytes
If increased glycolysis was the source of the energy to increase DNA repair and thus reduce DNA damage in UV-irradiated keratinocytes, then the inclusion of 2-deoxy-Dglucose (2DG) should impair DNA damage reduction, while the addition of oligomycin should have no effect. As previously observed, the addition of 24(OH)L 3 or 1,25(OH) 2 D 3 significantly reduced the CPDs (Figure 5A

Reductions in UV-Induced CPDs and 8-OHdG by 24(OH)L3 or 1,25(OH)2D3 Were Abolished in the Presence of the Glycolysis Inhibitor (2-Deoxy-D-glucose) but not by the Oxidative Phosphorylation Inhibitor (Oligomycin) in Human Primary Keratinocytes
If increased glycolysis was the source of the energy to increase DNA repair and thus reduce DNA damage in UV-irradiated keratinocytes, then the inclusion of 2-deoxy-D-glucose (2DG) should impair DNA damage reduction, while the addition of oligomycin should have no effect. As previously observed, the addition of 24(OH)L3 or 1,25(OH)2D3 significantly reduced the CPDs (Figure 5A,B) and 8-OHdG ( Figure 5C,D) in human primary keratinocytes after UV (p < 0.001). Treatment with 2DG abolished the reduction of UV-induced CPDs and 8-OHdG by 24(OH)L3 or 1,25(OH)2D3 measured 1.5 h after irradiation. Treatment with oligomycin did not affect the reduction of UV-induced CPDs or 8-OHdG with 24(OH)L3 or 1,25(OH)2D3 after 1.5 h. Treatment with the combination of 2DG and oligomycin also abolished the reduction of UV-induced photolesions in the presence of 24(OH)L3 and 1,25(OH)2D3 to a similar extent as 2DG alone ( Figure 5A-D).

Topical Treatment with 1,25(OH)2D3 or 24(OH)L3 Increased XPC and XPA in UV-Irradiated Skin Explants
If CPD repair by the nucleotide excision repair (NER) pathway were increased, it would be expected that an increase in key NER proteins such as XPC and XPA would be observed. In UV-irradiated, vehicle-treated skin explants, there was a small but not significant increase in XPC ( Figure 7A,B) or XPA ( Figure 7C,D), compared with non-irradiated skin explants. In contrast, significant increases in XPC (p < 0.01) ( Figure 7B) and XPA (p < 0.01) ( Figure 7D) were observed in skin explants treated topically with 1 × 10 −9 M 1,25(OH)2D3. Treatment with 1 × 10 −8 M 24(OH)L3 significantly increased both XPC and XPA (p < 0.05), but there was no significant increase in either protein after treatment with 1 × 10 −9 M 24(OH)L3 ( Figure 7B,D).

Topical Treatment with 1,25(OH) 2 D 3 or 24(OH)L 3 Increased XPC and XPA in UV-Irradiated Skin Explants
If CPD repair by the nucleotide excision repair (NER) pathway were increased, it would be expected that an increase in key NER proteins such as XPC and XPA would be observed. In UV-irradiated, vehicle-treated skin explants, there was a small but not significant increase in XPC ( Figure 7A,B) or XPA ( Figure 7C,D), compared with nonirradiated skin explants. In contrast, significant increases in XPC (p < 0.01) ( Figure 7B) and XPA (p < 0.01) ( Figure 7D) were observed in skin explants treated topically with 1 × 10 −9 M 1,25(OH) 2 D 3 . Treatment with 1 × 10 −8 M 24(OH)L 3 significantly increased both XPC and XPA (p < 0.05), but there was no significant increase in either protein after treatment with 1 × 10 −9 M 24(OH)L 3 ( Figure 7B,D).

Reductions of UV-Induced CPDs by 1,25(OH) 2 D 3 and 24(OH)L 3 Were Abolished by Knockdown of XPC or XPA
In order to see whether the NER proteins indeed were critical for the increased repair of CPDs, the keratinocytes were first transfected with small interfering RNA targeted to XPC mRNA (siXPC) or with a non-directed siRNA sequence (siCTRL), as described in [92]. XPC knockdown by siRNA was verified by Western blot ( Figure 8A). Human primary keratinocytes were irradiated 48 h after siRNA transfection and immediately treated with either vehicle or 1,25(OH) 2 D 3 or 24(OH)L 3 each at 1 × 10 −9 M for 3 h. Photomicrographs of immunohistochemical staining for UV-induced CPDs are presented in Figure 8B. The UVirradiated vehicle-treated cells showed a significant increase in CPDs compared with non-UV cells in cells transfected with a non-directed sequence (siCTRL) (p < 0.0001). Significant reductions in UV-induced CPDs were observed in cells treated with 1,25(OH) 2 D 3 and 24(OH)L 3 (p < 0.001 for both) in the siCTRL samples. Reductions of CPDs with 1,25(OH) 2 D 3 or 24(OH)L 3 were abolished in the presence of cells transfected with siRNA directed at XPC (siXPC) (Figure 8B,C).

Reductions of UV-Induced CPDs by 1,25(OH)2D3 and 24(OH)L3 Were Abolished by Knockdown of XPC or XPA
In order to see whether the NER proteins indeed were critical for the increased repair of CPDs, the keratinocytes were first transfected with small interfering RNA targeted to XPC mRNA (siXPC) or with a non-directed siRNA sequence (siCTRL), as described in [92]. XPC knockdown by siRNA was verified by Western blot (Figure 8A). Human primary keratinocytes were irradiated 48 h after siRNA transfection and immediately treated with either vehicle or 1,25(OH)2D3 or 24(OH)L3 each at 1 × 10 −9 M for 3 h. Photomicrographs of immunohistochemical staining for UV-induced CPDs are presented in Figure  A similar study was carried out in keratinocytes transfected with siRNA targeted to XPA mRNA (siXPA) or a non-directed siRNA sequence (siCTRL). Again, UV-irradiated A similar study was carried out in keratinocytes transfected with siRNA targeted to XPA mRNA (siXPA) or a non-directed siRNA sequence (siCTRL). Again, UV-irradiated vehicle-treated human primary keratinocytes showed significant increases in CPDs compared with non-irradiated cells in siCTRL samples ( Figure 8D,E; p < 0.0001). UV-irradiated keratinocytes treated with 1,25(OH) 2 D 3 or 24(OH)L 3 for 3 h had significantly reduced UV-induced CPDs (p < 0.001 or p < 0.01 respectively) in siCTRL samples. The reduction of CPDs by 1,25(OH) 2 D 3 or 24(OH)L 3 was abolished in cells transfected with siRNA directed at XPA (Figure 8D,E).

Discussion
The treatment with 1,25(OH) 2 D 3 has been shown to reduce UV-induced DNA damage in skin in many studies [52,55,57,76,79,85,[93][94][95]. The current study investigated the photoprotective properties of one of the most abundant "over-irradiation" metabolites, 24(OH)L 3 , in comparison with 1,25(OH) 2 D 3 . This study tested only 24(OH)L 3. While further metabolism of 24(OH)L 3 by other CYP enzymes known to metabolize vitamin-D-related compounds is theoretically possible, there is no precedent for CYP24A1, which metabolizes 25-hydroxyvitamin D and 1,25(OH) 2 D 3 to their 24-hydroxylated derivatives, to act on a sterol with an intact B-ring. Even though it is possible that CYP27A1 acts on 24(OH)L3, as it can act on other lumisterol compounds (R. Tuckey, personal communication), it is doubtful that there is enough CYP27A1 in skin to make a difference. Whether any metabolism of 24(OH)L3 by CYP27A1 changes biological activity is also unknown. UV-induced CPDs, as well as 8-OHdG, were reduced in a concentration-dependent manner by the addition of 24(OH)L 3 immediately after UV exposure, similar to 1,25(OH) 2 D 3 . In keratinocytes, both compounds were effective at a low concentration of 1 × 10 −10 M, but in human skin explants, the minimal effective dose of 1,25(OH) 2 D 3 to reduce both CPDs and 8-OHdG was 10-fold higher at 1 × 10 −9 M. The minimal effective dose of 24(OH)L 3 in skin explants was a further 10-fold higher. It is likely that the thickness of epidermis in the human skin reduced the penetration of both compounds, with that of 24(OH)L 3 reduced to a greater extent than that of 1,25(OH) 2 D 3 , although this is yet to be investigated in more detail.
Our results indicated that at least part of the reason for the reduction in DNA damage with 24(OH)L 3 was an increase in DNA repair as evidenced by the increased intensity and number of UDS-positive nuclei in the presence of 24(OH)L 3 compared with the UV vehicle in UV-exposed keratinocytes. These increases were similar to those seen with 1,25(OH) 2 D 3 . As illustrated in Figure 3A,B, DNA repair was observed mainly in the nuclei of keratinocytes, although the repair of mitochondrial DNA cannot be entirely excluded. We have previously reported increased DNA repair after UV with 1,25(OH) 2 D 3 [79]. DNA repair is an energy-intensive metabolic process. UV radiation damages mitochondria, and the damage impairs their main function, cellular respiration [31,96,97]. An earlier study by our group indicated that oxygen consumption rates, which are measures of oxidative phosphorylation, decreases after UV exposure [79]. As shown here, oligomycin, an inhibitor of oxidative phosphorylation [88,89], had no effect on the ECAR response of the UV exposed cells due to inactive oxidative phosphorylation. As expected, the treatment of the keratinocyte cultures with oligomycin also had no effect on the reduction of UV-induced DNA damage with 24(OH)L 3 or the hormonal form of vitamin D.
Although ATP levels were reduced after UV, treatment with either 1,25(OH) 2 D 3 or 24(OH)L 3 increased ATP. We looked at an alternative source of energy, glycolysis. There were no significant changes in glycolysis, as measured by ECAR in UV-irradiated cells injected with vehicle. In contrast, glycolysis was increased by both 1,25(OH) 2 D 3 and 24(OH)L 3 in UV-irradiated keratinocytes, as reported previously for 1,25(OH) 2 D 3 [79]. Oligomycin increased ECAR in non-irradiated cells, indicating that oxidative phosphorylation was still intact in these cells, unlike the UV-exposed cells, as energy production in the cell switched from oxidative phosphorylation to glycolysis upon the addition of the oxidative phosphorylation inhibitor in the non-irradiated keratinocytes. As seen in Figure 4A, the addition of 2-deoxy-D-glucose suppressed glycolysis/ECAR in all keratinocytes. The addition of 2-deoxy-D-glucose to the keratinocytes also abolished the reduction in UVinduced CPDs and 8-OHdG by 1,25(OH) 2 D 3 or 24(OH)L 3 , consistent with the proposal that the active glycolysis produced by treatment with 1,25(OH) 2 D 3 or 24(OH)L 3 was critical for the reduction in UV-induced DNA damage.
The electron transport chain in mitochondria is the main source of intrinsic ROS production in cells [98]. UV irradiation increases ROS production, and the damage to mitochondria by UV may further increase cellular ROS levels [99,100]. Increased ROS levels were observed in UV-irradiated keratinocytes at 15 min and 1.5 h post-UV. The UV-irradiated keratinocytes treated with 1,25(OH) 2 D 3 or 24(OH)L 3 had significantly reduced ROS, indicating that these compounds could contribute to a reduction of ROS levels after UV. While one explanation could be just that 1,25(OH) 2 D 3 or 24(OH)L 3 slowed oxidative phosphorylation, we have previously reported that the treatment of keratinocytes with 1,25(OH) 2 D 3 increased an early form of mitophagy [101], leading to mitochondrial repair [79] and reduced ROS. There are data indicating that 1,25(OH) 2 D 3 via the vitamin D receptor (VDR) is able to bind mitochondrial DNA and affect the transcription of oxidative phosphorylation subunits [102], while silencing of the VDR in various cell lines caused excessive ROS generation [103]. There is also evidence that 1,25(OH) 2 D 3 activates the Nrf2-Keap1 antioxidant pathway, which ameliorates oxidant stress [104][105][106], and that 1,25(OH) 2 D 3 , lumisterol, and even tachysterol hydroxyderivatives do so in UV-exposed keratinocytes [107,108] and thus potentially reduce oxidative DNA damage. It is highly likely that reduced ROS levels as a result of treatment with 1,25(OH) 2 D 3 or 24(OH)L 3 directly contribute to reduced 8-OHdG levels [91] and nitrosative stress [109] in UV-irradiated keratinocytes. Importantly, with reduced ROS and reactive nitrogen species [53,76], there will be less damage to cellular proteins, including proteins involved in DNA repair, and thus more effective repair [34,110] Since the abovementioned mechanisms may collectively contribute to the increased DNA repair after UV, we explored two proteins involved in the nucleotide excision repair pathway for CPDs. XPC is an initial damage recognition protein in the GG-NER pathway, while XPA helps to verify the DNA damage and stabilize the DNA as it is repaired and is required for both GG-NER and TC-NER [111]. XPC and XPA accumulate at the sites of DNA damage [112]. UV-induced CPDs cause only a small distortion in the DNA helix and so are dependent for detection on the binding of XPE, a complex of damaged DNA binding proteins 1 and 2 (DDB1 and DDB2) [38]. DDB recruits XPC, which together with XPA is required for the successful initiation of NER [113]. In the current study, the XPC and XPA protein levels were increased in response to treatment with the lumisterol-derived 24(OH)L 3 as well as 1,25(OH) 2 D 3 in UV-irradiated human explants. Furthermore, knockdown of either XPC or XPA abolished the reduction in CPDs normally seen after the addition of either 1,25(OH) 2 D 3 or 24(OH)L 3 , indicating key roles for these proteins in this process. No other changes in DNA repair proteins after 1,25(OH) 2 D 3 or 24(OH)L 3 treatment were examined in this study. It has previously been reported that the treatment of human keratinocytes in culture with 1,25(OH) 2 D 3 upregulated mRNA for XPC and DDB2 [114]. In an earlier study, both XPC and DDB2 protein levels were significantly increased in human skin biopsies treated topically with 1,25(OH) 2 D 3 at non-UV or UV-irradiated sites [115].
A possible mechanism for the increase in XPC involves phosphatase and tensin homolog deleted on chromosome ten (PTEN). PTEN is tumor suppressor and acts as a negative regulator of the oncogenic acutely transforming retrovirus AKT8 in the rodent Tcell lymphoma (AKT) pathway [116]. It has been reported that UVB down-regulates PTEN in primary human keratinocytes and in mouse skin in an AKT-and extracellular-signalregulated kinase (ERK) 1/2 -dependent manner [117]. This downregulation of PTEN reduces global genomic NER, through reductions in XPC [118]. More recent studies by Shariev et al. showed that exposure to solar-simulated UV decreased PTEN levels in Skh:hr1 hairless mouse skin [119], as well as primary melanocytes and melanoma cells [120]. The addition of 1,25(OH) 2 D 3 inhibited this UV-induced depletion of PTEN [119], which likely explains the suppression of AKT phosphorylation after UV by 1,25(OH) 2 D 3 [79].
In all this, although vitamin D 3 and lumisterol hydroxyderivatives have been shown to bind to retinoid receptors [71] and to liver X receptors [121], the vitamin D receptor (VDR) in skin and the endoplasmic reticulum protein p57 (ERp57) are clearly key for the reductions in DNA damage by 1,25(OH) 2 D 3 or 24(OH)L 3 . The reductions in both CPDs and 8-OHdG after UV by either 1,25(OH) 2 D 3 or 24(OH)L 3 , as well as a CYP11A1 derivative of vitamin D 3 , 20(OH)D 3 , were abolished with the knockdown of either ERp57 or VDR [85,122]. VDR-null mice show increased UV-induced DNA damage [123], as well as increased skin tumor formation after UV or chemical carcinogenesis [123,124]. VDR mRNA expression and protein levels were reduced in UVB-irradiated keratinocytes [125]. When human keratinocytes were treated with 24(OH)L 3 , VDR mRNA and protein levels increased in both UV-irradiated and non-irradiated cells [71].

Conclusions
These data collectively indicated that the major over-irradiation metabolite, 24(OH)L 3 , derived from lumisterol through the actions of CYP11A1 in skin, contributed to the reduction of UV-induced damage in a manner similar to the vitamin D hormone, 1,25(OH) 2 D 3 , in keratinocytes and the whole skin. Other CYP11A1 derivatives produced in skin from both vitamin D 3 and lumisterol [68,126,127] also reduced UV-induced DNA damage [71,122,126,128]. These findings are consistent with the proposal that this range of UV-generated, 7-dehydrocholesterolderived, CYP11A1-produced compounds also help reduce UV-induced DNA damage, which is a key precursor to skin tumor development. This proposal would provide an explanation for the reported observations that mice with a knockout of CYP27B1, which is solely responsible for the production of 1,25(OH) 2 D 3 and so cannot produce 1,25(OH) 2 D 3 , nevertheless are not more susceptible to UV-induced skin tumors than wild-type mice [129], whereas VDR knockout mice show much greater susceptibility to UV-induced photocarcinogenesis [123].